Membrane fractions of 1,2-sn-diacylglycerol-enriched cells

ABSTRACT

The invention concerns membrane fractions of cells containing a recombinant MGDG synthase and enriched with 1,2-sn-diacylglycerol, their preparation method, their use for screening molecules inducing MGDG synthase activity and a method for screening molecules inducing MGDG synthase activity using said membrane fractions.

[0001] The present invention relates to membrane fractions of cellscontaining a recombinant monogalactosyldiacylglycerol (MGDG) synthaseand enriched in 1,2-sn-diacylglycerol (DAG), to the method of preparingthem, to their use for screening molecules having an effect on MGDGsynthase activity and to a method of screening molecules having aneffect on MGDG synthase activity, using these membrane fractions.

[0002] MGDG is known to be in all plasts analyzed to date: it is themost abundant lipid of plastid membranes, where it represents more than50% of glycerolipids. MGDG is vital to plast biogenesis and to cellsurvival, and does not exist in the other membrane systems, inparticular in animal cells (Douce, Sciences, 1974, 183, 852-853); thebiosynthesis thereof is catalyzed in the envelope by a uridine5′-diphosphate galactose (UDP-gal) 1,2-diacylglycerol3-β-D-galactosyltransferase (EC 2.4.1.46) also called MGDG synthase,according to the following reaction:

[0003] MGDG synthase is a bifunctional enzyme which binds substrates ina non-ordered manner (Maréchal et al., J. Biol. Chem, 1994, 269,5788-5798), which has oxidation-sensitive cysteines and which isimportant for catalysis (Maréchal et al., J. Biol. Chem., 1995, 270, 11,5714-5722).

[0004] MGDG synthase is therefore also an enzyme essential to plastbiogenesis and is, consequently, a target of choice for selecting orscreening molecules with herbicidal potential. In addition, theparasites responsible for malaria (4 species of Plasmodium, including P.falciparum), for toxoplasmosis (Toxoplasma gondii) and for scourges ofthe veterinary field, such as coccidiosis (Eimeria) contain degeneratechloroplasts (apicoplasts) which have been demonstrated to be essentialto parasite survival (G. McFadden and David Roos, “Apicomplexan plastidsas drug targets”, 1999, 7, No. 8, 328-333).

[0005] The parasites which contain these apicoplasts are calledapicomplexan parasites.

[0006] A molecule which has an inhibitory action on MGDG synthaseactivity therefore has a high herbicide and anti-apicomplexan parasitepotential and can be used advantageously as a novel medicinal producteffective against said apicomplexan parasites or as a herbicide.

[0007] The use of an MGDG synthase for selecting or screening productsinhibiting MGDG synthase activity, able to be used as herbicides or asactive principles against apicomplexan parasites has already beenproposed, in particular in patent application FR-A-2 790 915.

[0008] According to that application, the selection and/or screening ofsuch products is (are) carried out according to a method comprisingincubating a test substance with an MGDG synthase embedded in biologicalmembranes, and then measuring the specific enzyme activity, after saidincubation.

[0009] The biological membranes used in that prior application can inparticular be plastid membranes isolated from plants or else membranefractions of E. coli overexpressing a recombinant MGDG synthase.

[0010] Measuring the galactosylation activity carried out by MGDGsynthase is, at the current time, very complex and cannot be readilyminiaturized, in particular for the following reasons:

[0011] (1) Addition of two substrates: The two substrates of the enzyme(DAG and UDP-gal) should be added simultaneously to the incubationmedium. Homogeneity of the system is a problem, in particular due to thefact that these two substrates have very different physicochemicalproperties: one is very hydrophilic (UDP-gal), the other is veryhydrophobic (DAG). The control of the introduction of these twosubstrates is therefore difficult to miniaturize.

[0012] (2) Addition of a detergent: DAG is so hydrophobic that it is notwater-miscible. In order for the enzyme to have access to this exogenoussubstrate, a detergent therefore has to be introduced into theincubation medium, for example3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whichpulverizes the biological membranes and allows a rearrangement of allthe hydrophobic molecules in the form of micelles. The micelles are toosmall to be separated from the reaction medium by filtration orcentrifugation (Stoke's radius 3.8 nm; Marechal et al, 1994, mentionedabove).

[0013] (3) Phase separation: In order to extract the lipid phasecontaining the reaction product (MGDG), a mixture of organic solvents(chloroform and methanol) is added at the end of the reaction accordingto the method described by Bligh et al. (Can. J. Biochem. Physiol.,1959, 37, 911-917). A biphase forms and the lipids are recovered in thelower organic phase. The formation of a biphase and the extraction of anorganic phase are processes which are too sophisticated to be used in aminiaturized process.

[0014] Now, in the context of the search for molecules having an effecton MGDG synthase activity, it is essential to have a simple, relativelyinexpensive, rapid and miniaturizable method for testing a very largenumber of molecules potentially able to be used as herbicides or asactive principles against apicomplexan parasites.

[0015] The inventors have developed the subject of the invention inorder to remedy these problems.

[0016] Specifically, the inventors have developed new biologicalmembrane fractions containing, in the same lipid leaflet, both MGDGsynthase and DAG. These membranes can be used in an enzyme method forautomated high throughput screening (HTS) of molecules having an effecton MGDG synthase activity (inhibitors or activators).

[0017] A subject of the present invention is therefore plasma membranefractions from prokaryotic cells or eukaryotic animals, consisting of alipid leaflet containing at least one recombinant MGDG synthase,characterized in that said fractions contain at least 1% by weight ofDAG relative to the total weight of protein, and in that they are in theform of spherical vesicles.

[0018] The inventors have in fact demonstrated that the simultaneouspresence of MGDG synthase and DAG in said membrane fractions makes itpossible to use these membrane fractions in a method of screening and/orselecting molecules having an effect on MGDG synthase activity, in whichthe volumes of the reaction media can be considerably reduced, thusenabling miniaturization of said method.

[0019] By virtue of these membrane fractions, the addition of detergentto promote the control MGDG synthase/DAG mixing is eliminated, and theenzyme reaction takes place directly within the membranes.

[0020] According to a preferred embodiment of the invention, the MGDGsynthase/DAG molar ratio is less than 10, and even more particularlyless than 0.12.

[0021] Specifically, the enzyme/substrate ratio should respect theconditions for measurement making it possible to use theMichaelis-Menten enzymological model; in particular, the substrateshould not be the limiting factor of the initial reaction.

[0022] The membrane fractions in accordance with the invention arepreferably in the form of spherical vesicles made up of a lipid bilayer.

[0023] These membrane vesicles are generally between 0.1 μm and 10 μm insize.

[0024] Within these vesicles, the MGDG synthase is, in general, locatedon the inner face of the lipid bilayer.

[0025] These vesicles may be in the form of noninverted, inverted orhybrid vesicles.

[0026] Disruption of the membranes in fact allows the formation ofinverted or noninverted vesicles; but in both cases, the MGDG synthaseis on the face opposite most of the DAG. Fusion between inverted andnoninverted vesicles can generate a new family of vesicles having theMGDG synthase and the DAG in the same lipid leaflet.

[0027] This type of hybrid vesicle can exhibit increased catalyticactivity (accessible substrate) and is, consequently, preferredaccording to the invention.

[0028] A subject of the invention is also a method of preparing membranefractions as described above, characterized in that it consists:

[0029] in a first step, in transforming prokaryotic cells or eukaryoticanimals with a construct containing the gene encoding a plant MGDGsynthase,

[0030] in a second step, in culturing said cells in a culture mediumwhich promotes protein synthesis, so as to induce the synthesis of MGDGsynthase by said cells,

[0031] in a third step, in incubating the cells cultured in thepreceding step in a reaction medium containing at least onephospholipase C,

[0032] and then, in a fourth step, in fractionating the cells thusenriched in DAG so as to obtain membrane fractions in the form ofspherical vesicles containing at least one recombinant MGDG synthase andat least 1% by weight of DAG relative to the total weight of proteins ofsaid membrane fractions.

[0033] Among the prokaryotic cells which can be used according to thismethod, mention may be made of bacteria such as E. coli, which areparticularly preferred according to the invention.

[0034] Among the eukaryotic cells which can be used according to thismethod, mention may be made of cells from yeast, such as Saccharomycescerevisiae, cells from insects, such as drosophila, and also mammaliancells conventionally used to express genes, such as COS cells and CHOcells.

[0035] The transformation of the cells in the first step is preferablycarried out on bacterial cells, and more particularly on E. coli cells.

[0036] This transformation can be carried out according to the methoddescribed in patent application FR-A-2 790 915, for example by heatshock, with a plasmid pET-Y3a containing the sequence encodingArabidopsis thaliana MGDG synthase A (Miege et al., Eur. J. Biochem.,1999, 265, 990-1001).

[0037] The culture medium used in the second step is a rich culturemedium, in order to promote expression of the MGDG synthase, and ischosen as a function of the type of cells to be cultured. In theparticular case of bacterial cells, and in particular of E. coli, LuriatBroth (LB) medium can be used.

[0038] The use of PLC during the third step of the method of preparationin accordance with the invention is essential to the enrichment of theplasma cell membranes in DAG.

[0039] Specifically, these cell membranes are rich in phospholipids,particularly in phosphatidylethanolamine (80% of phospholipids). Thesephospholipids can be hydrolyzed by PLC, which is not specific for thepolar head.

[0040] PLC catalyzes the conversion of phosphatidylethanolamine to DAGaccording to the following reaction:

[0041] PLC therefore makes it possible to enrich the cell membranes inDAG by hydrolysis of its phospholipids.

[0042] The nature of the PLC used according to the invention is notcritical, on condition that it is active on the phospholipids of themembranes intended to be enriched in DAG.

[0043] According to a particularly preferred embodiment of theinvention, a Bacillus cereus phospholipase C is used.

[0044] The PLC is preferably used at a concentration between 1 U and 20U per ml of reaction medium, and even more preferentially at aconcentration between 5 and 12 U/ml.

[0045] Of course, the reaction medium used in the third step isgenerally a buffer medium the pH of which should be compatible with thecorrect functioning of the PLC. This pH is generally between 6 and 8.

[0046] In the fourth step, the membranes are fractionated so as togenerate membrane fractions which close up spontaneously in the form ofmembrane vesicles. The fractionation of the membranes can be carried outby mechanical shock, for example using a French press, by thermal shock(freezing/thawing), or by osmotic, electric or else physical shock, suchas by sonication.

[0047] The membrane fractions thus obtained can then optionally bepurified, for example on a cushion of Percoll.

[0048] When the preparation of the membrane fractions in accordance withthe invention is finished, they can optionally be frozen before beingused for selecting or screening molecules having an effect on MGDGsynthase activity.

[0049] A subject of the invention is therefore also the use of themembrane fractions as described above, for selecting and/or screeningmolecules having an effect on MGDG synthase activity.

[0050] In particular, a subject of the invention is the use of themembrane fractions as described above, for selecting or screeningmolecules inhibiting MGDG synthase activity, able to be used as activeprinciples against parasites or as herbicides.

[0051] A subject of the invention is also a method of selecting and/orscreening molecules having an effect on MGDG synthase activity,characterized in that it comprises:

[0052] a step comprising incubation of the test substance(s) with asufficient amount of membrane fractions as defined above and ofradiolabeled UDP-galactose in an aqueous phase having a pH of between 4and 11,

[0053] a step comprising washing of the membrane fractions,

[0054] a step comprising separation of the membrane fractions,

[0055] then a step comprising determination of the MGDG synthaseactivity.

[0056] According to this method, the enzyme reaction takes placedirectly within the membrane fractions, the size of which is compatiblewith separation from the reaction medium by centrifugation or byfiltration. Using this means, it is no longer necessary to use organicsolvent to extract the reaction products, the MGDG in fact being trappedin the membrane fractions on which a simple measurement of theradioactivity of the radiolabeled galactose incorporated can be carriedout.

[0057] According to a preferred embodiment of the invention, the aqueousincubation phase contains a buffer and has a pH of between 6 and 8.

[0058] By way of example, the buffer may in particular be3-(N-morpholino)propanesulfonic acid or a KCl/K₂HPO₄ mixture.

[0059] The amount of UDP-galactose to be used in the incubation mediumshould, of course, be sufficient so as not to constitute a limitingfactor of the reaction. This amount is preferably between 0.1 and 10nmol per μl of reaction medium.

[0060] The incubation step is preferably carried out at ambienttemperature, for a period of at least 10 seconds, and even moreparticularly for a period of between 1 and 45 minutes.

[0061] At the end of the incubation step, the reaction is preferablystopped by cooling the incubation medium (in general to a temperature ofapproximately 4° C.) or by centrifugation.

[0062] When the reaction is finished, the step comprising washing of themembrane fractions is carried out in order to eliminate the excessradiolabeled UDP-galactose which has not been incorporated by themembrane fractions. One or more successive washes may be carried out,generally with water.

[0063] The membrane fractions are then separated by centrifugation or byfiltration, the latter technique being particularly suitable forminiaturization of the method on microplates.

[0064] The determination of the MGDG synthase activity is carried out bymeasuring the amount of radiolabeled galactose incorporated into themembrane fractions. This measurement is conventionally carried out usinga radioactivity counter.

[0065] The method of selecting and/or screening molecules having aneffect on MGDG synthase activity in accordance with the invention can bereadily miniaturized since it uses small reaction volumes and does notuse detergents or organic solvent, as is the case in the methodspreviously described in the prior art.

[0066] A subject of the invention is also therefore microtitrationplates comprising a multitude of wells, characterized in that the bottomof the wells consists of a filter and in that said wells containmembrane fractions as described above.

[0067] These microplates can be stored in a freezer before use. They canoptionally be equipped with a detachable bottom.

[0068] The microplates in accordance with the invention make it possibleto determine the MGDG synthase activity by direct measurement of theradioactivity of the radiolabeled galactose incorporated into themembrane fractions retained by the filter at the bottom of the wells.

[0069] They may therefore be used to test large “chimiotheques”[chemical libraries] of molecules for their activity of an effect onMGDG synthase activity.

[0070] Finally, a subject of the invention is the use of at least onemolecule inhibiting MGDG synthase activity as selected in accordancewith the method of screening and selecting in accordance with theinvention, for preparing an antiparasitic medicinal product or aherbicide.

[0071] Besides the arrangements above, the invention also comprisesother arrangements which will emerge from the following description,which refers to an example of preparation of membrane fractions inaccordance with the invention, to a comparative example of determinationof MGDG synthesis activity, to an example of demonstration of thepresence of MGDG in an apicomplexan parasite, and to the attachedfigures, in which:

[0072]FIG. 1 represents the number of nmole of galactose incorporatedper hour into membrane fractions enriched in DAG as a function of thenumber of μg of proteins,

[0073]FIG. 2A represents the number of nmole of galactose incorporatedper hour and per mg of proteins (membrane fractions enriched in DAG) asa function of the number of μmole of DAG,

[0074]FIG. 2B represents an inverse coordinate plot of the Line Weaverand Burk type on which the inverse of the number of μmoles of galactoseincorporated per hour and per mg of proteins is expressed as a functionof the inverse of the number of μmoles of DAG;

[0075]FIG. 3 represents the amount of labeled galactose incorporated bythe membrane lipids of Toxoplasma gondii.

[0076] It should be clearly understood, however, that these examples aregiven only by way of illustration of the subject of the invention, ofwhich they in no way constitute a limitation.

EXAMPLE 1 Preparation of Membrane Fractions Containing an MGDG Synthaseand DAG

[0077] 1) Production of Recombinant MGDG Synthase in E. coli

[0078] A—Transformation of the Bacteria

[0079] The bacteria are transformed according to the method described inpatent application FR-A-2 790 915.

[0080] All the cultures are prepared under sterile conditions. Competentbacteria (bacterial strain BL21 or BLR of Escherichia coli) aretransformed by heat shock with a plasmid pET-Y3a which makes it possibleto overcome the problem due to the fact that the deduced sequence of theMGDG synthase contains 22 arginine residues, among which 17 are encodedby AGG or AGA, codons which are in fact used very little in E. coli.

[0081] The plasmid pET-Y3a has been described in patent applicationFR-A-2 790 915 and is constructed by inserting the arg U gene (or DNA Y)encoding the arginine transfer RNA associated with the rare codonsAGA/AGG, into the plasmid pET-3a (Novagen). The plasmid pET-Y3a containsthe sequence encoding Arabidopsis thaliana MGDG synthase A (under thecontrol of a promoter inducible withisopropyl-p-D-thiogalactopyranoside: IPTG), a carbenecillin resistancegene and a sequence encoding the arginine transfer RNA. This ARG4 tRNAallows the synthesis of proteins such as MGDG synthase, the sequence ofwhich contains many Arg codons, which are rare in bacteria.

[0082] B—Production of Recombinant MGDG Synthase A in E. coli

[0083] A colony isolated from recombinant bacteria is transferred into 8ml of Luria Broth (LB) medium in the presence of antibiotic (20 μg/mlfinal concentration of carbenecillin). The preculture is incubated at37° C., with regular shaking, and the evolution of bacterial growth isfollowed by measuring the optical density (OD) at 600 nm, until a valueof 0.5 is obtained.

[0084] The preculture is transferred into 500 ml of LB medium (20 μg/mlfinal concentration of carbenecillin) and incubated at 37° C. withregular shaking. At an OD measured at 600 nm of 0.5, the bacterialpopulation is then in its exponential growth phase, which is a time ofintense protein synthesis.

[0085] The addition of IPTG (0.4 mM final concentration) makes itpossible to induce synthesis of the recombinant MGDG synthase.

[0086] The culture is then incubated for 3 hours with shaking at 28° C.in order to promote production of the protein in its active form.

[0087] The suspension of induced bacteria is divided up into twofractions of 250 ml and subjected to centrifugation for 15 minutes at 5000 rpm (Sorvall® RCSC centrifuge, GS-3 rotor).

[0088] The recovered pellets are resuspended in 10 ml of culture medium(LB, carbenecillin at 20 μg/ml final concentration) and centrifuged for15 minutes at 5 000 rpm (Sorvall® RCSC, SLA 600 TC rotor).

[0089] The supernatant is removed and the bacterial pellet is stored at−80° C.

[0090] 2) Enrichment of the Bacterial Membranes in DAG by Treatment withPhospholipase C.

[0091] 5 ml of pellet of the bacteria induced for MGDG synthase in theprevious step are resuspended in 5 ml of 10 mM3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.8, 1 mM dithiothreitol(DTT) and 10% (w/v)glycerol.

[0092] 20 μt of Bacillus cereus phospholipase C (PLC)(phosphatidyl-choline cholinephosphohydrolase, EC 3.1.4.3) are added inorder to obtain a final concentration of 8 U/ml. The reaction is carriedout at ambient temperature for 3 hours with stirring and is stopped byadding EDTA (0.4 M final concentration).

[0093] 3) Purification of Bacterial Membranes

[0094] A solution of the induced bacteria, the membranes of which areenriched in DAG by treatment with PLC, as obtained above in the previousstep, is taken up in 50 ml of 5 mM MOPS, pH 7.8, 0.5 mM DTT, 5%(w/v)glycerol.

[0095] The bacteria are ruptured at high pressure (800 psi) using aFrench press: the membranes in aqueous solution then organize themselvesspontaneously into vesicles and into inverted vesicles.

[0096] 6 ml of the lysate obtained are deposited on a cushion of 35%Percoll (in 10 mM MOPS, pH 7.8, 1 mM DTT, 10% glycerol) and thencentrifuged for 10 minutes at 5 000 rpm, at a temperature of 4° C.(Sorvall® RC SC, HB-6 rotor).

[0097] Vesicles consisting of a lipid bilayer containing MGDG synthaseand at least 1% by weight of DAG relative to the total weight of proteinare obtained.

[0098] Four fractions are then formed: a supernatant (in the upper partof the tube), the supernatant/Percoll interface, the cushion of 35%Percoll and a pellet.

[0099] All the membrane fractions are collected and washed by dilutionwith 5 volumes of 10 mM MOPS, pH 7.8, 1 mM DTT, 10% (w/v) glycerol, andcentrifuged for 15 minutes at 5 000 rpm at a temperature of 4° C.(Sorvall® RCSC, HB-6 rotor).

[0100] The supernatants are removed and the pellets, which are fragile,are carefully taken up in 1 ml of washing medium so as to be centrifugedagain for 10 minutes at 13 000 rpm at a temperature of 4° C. (Eppendorffcentrifuge 5804).

[0101] The pellets are resuspended in 500 μl of 10 mM MOPS, pH 7.8, 1 mMDTT, 10% (w/v)glycerol and are stored at −20° C.

EXAMPLE 2 Comparative Determination of MGDG Synthesis Activity Accordingto the Prior Art and According to the Invention

[0102] I—Conventional measurement of the MGDG synthesis activityaccording to the method of Bligh and Dyer (1959)

[0103] This method was described in the article by Bligh and Dyer, “ARapid Method Of Total Lipid Extraction and Purification”, Can. J.Biochem. Physiol., 1959,37,911-917.

[0104] The measurement of the galactosylation activity is based onincorporation of the galactose originating from the radioactive (¹⁴C)UDP-gal into the lipid fraction of the reaction medium.

[0105] The reaction is carried out at ambient temperature. 100 μg of DAGhydrophobic substrate (1 mg/ml) and 200 μg of phosphatidylglycerol (PG)in solution in chloroform (10 mg/ml) are introduced into a tube, driedunder argon and then resuspended with 11 pi of a detersive medium (85 mMCHAPS, 0.7 M MOPS, 14 mM DTT), 75 μl of KCl (1 M) and 75 μl of KH₂PO₄ (1M).

[0106] The presence of the detergent, in this case CHAPS, makes itpossible to create mixed micelles containing the detergent, the DAG, thePG and the MGDG synthase originating from the sample.

[0107] After addition of 150 μl of sample, the final composition of thereaction medium (50 mM MOPS, 1 mM DTT, 250 mM KCl, 250 mM KH₂PO₄, 6 mMCHAPS in 300 μl of final volume) satisfies the criteria of the surfacedilution model (Marechal et al, 1995 mentioned above).

[0108] This example uses CHAPS as detergent, but it is also possible touse other detergents such as, for example, cholate, deoxycholate,Triton-X100®, NONIDET®, octylglucoside or lauryldimethylamine oxide(LDAO).

[0109] The reaction is started by introducing 10 μl ofUDP-[¹⁴C]-galactose (New England Nuclear 25 Bq/μmol, 10 mM). Thereaction is stopped by adding 1.5 ml of a 1/2 (v/v) chloroform/methanolmixture.

[0110] The addition of 0.5 ml of chloroform and of 0.6 ml of water makesit possible, after centrifugation for 10 minutes at 1000 rpm (EppendorffA-4-44 centrifuge, 5804 rotor), to obtain a distinct biphase (a highlyradioactive aqueous phase in the upper part of the tube, and an organicphase in the lower part).

[0111] The aqueous phase contains the radioactivity of the residualUDP-gal not used by the enzyme, while the organic phase contains theradioactivity of the hydrophobic product of the reaction: the MGDG.

[0112] After two washes of the aqueous phase with an identical volume ofaqueous phase without UDP-[¹⁴C]-gal, the organic phase containing theMGDG produced is transferred into a counting vial.

[0113] The fraction recovered is dried under argon and taken up in 10 mlof scintillation fluid, and the radioactivity of the sample isestimated.

[0114] The galactosylation activity is defined by the number of μmol ofgalactose incorporated into the lipid fraction per mg of protein and perhour.

[0115] II—Measurement by purification of the membranes aftercentrifugation in accordance with the invention.

[0116] This measurement can be carried out only for a sample consistingof vesicles of membranes sequestering both the enzyme (MGDG synthase)and its hydrophobic substrate (DAG). Then only in this case, it is nolonger necessary to add a detergent so that enzyme and substrate comeinto contact.

[0117] The reaction medium used is different from that given in theconventional method after extraction of the lipids with organicsolvents.

[0118] The membrane fraction sample containing the MGDG synthase and theDAG, as obtained above in example 1, step 3, is suspended in a finalvolume of 300 μl containing 250 mM KCl and 250 mM KH₂PO₄.

[0119] The reaction is started by adding UDP-radiolabeled [¹⁴C]gal.

[0120] The reaction is stopped by transferring to ice for 10 minutes.

[0121] Washing of the excess UDP-[¹⁴C]gal (not incorporated into themembranes) is carried out by centrifugation of the sample for 10 minutesat 13 000 rpm at a temperature of 4° C., and taking up the pellet in 500μl of sterile water.

[0122] This washing step is repeated three times. The pellet obtained isdried by centrifugation under vacuum for 1 hour (Eppendorff Concentrator5301 Speed-vac). The dry sample is then taken up in a ½ (v/v)chloroform/methanol mixture, in order to transfer it into a countingvial, dried under argon, and solubilized with 10 ml of scintillationfluid.

[0123] The radioactivity of the sample is estimated using a Kontron®(Betamatic) counter.

[0124] The galactosylation activity is defined by the number of μmole ofgalactose incorporated per mg of protein and per hour.

[0125] III—Polyacrylamide Gel Electrophoresis Under DenaturingConditions

[0126] The samples to be analyzed are suspended in the loading medium(0.15 M Tris HCl, pH 6.8, 10% glycerol, 0.02% SDS, 0.01% bromophenolblue, 0.025% DTT), and are then boiled for 4 minutes.

[0127] The acrylamide solutions are prepared in a 25 mM Tris buffer (pH8.3 in the separating gel and pH 6.5 in the stacking gel) containing0.192 M glycine and 0.1% sodium dodecyl sulfate (SDS).

[0128] The electrophoresis on a stacking gel (5% acrylamide) and then ona separating gel (12% acrylamide) is carried out at ambient temperaturein a 25 mM Tris buffer containing 0.192 M glycine (pH 8.3) and 0.1%(w/v) SDS (U.K. Laemmli, Nature, 1970, 227, 680-683), under a constantvoltage of 100 V.

[0129] The migration is stopped when the bromophenol blue leaves thegel.

[0130] The proteins are then stained with Coomassie blue (0.5% (w/v)Coomassie brilliant blue 8250, 25% methanol, 10% (v/v) acetic acid).

[0131] The gel is destained with successive baths of 25% isopropanol,10% (v/v) acetic acid.

[0132] IV—Assaying of Proteins

[0133] Principle: the proteins are assayed by the Lowry method (Lowry etal., J. Biol. Chem., 1951, 193, 265-275), by measuring two simultaneouscolored reactions.

[0134] A first reaction similar to the “biuret” reaction leads to theformation of a complex between the peptide bonds of the proteins(—CO—NH—) and the Cu²⁺ ions in alkaline medium, and a second reactionleads to reduction of the Folin-ciocalteu reagent by the phenols of thetyrosines. This method makes it possible to assay solutions having aconcentration ranging from 2 to 200 mg/ml.

[0135] Experimental approach: the volume of the sample to be assayed isadjusted to 200 μl with sterile water then 1 ml of assay reagentprepared extemporaneously (50 volumes of 2% CO₃Na₂, 0.1 N NaOH+1 volumeof CuSO₄, 0.5% SH₂O, 1% sodium tartrate).

[0136] After reaction for 10 minutes at 20° C., 100 μl ofFolin-ciocalteu reagent are added and the reaction is incubated at 20°C. for 30 minutes.

[0137] The amount of proteins in the sample to be assayed is determinedby comparison of absorbence at 750 nm with a standard range establishedwith bovine serum albumin (BSA).

[0138] V—RESULTS

[0139] A—The Recombinant Bacteria must be Artificially Enriched in DAGin Order to Allow Measurement of the Galactosylation Activity in thePresence of UDP-Gal

[0140] Analysis of the total proteins of bacterial samples taken duringthe induction step (example 1, step 1, FIG. 1) shows that induction withIPTG leads to an accumulation of MGDG synthase A representingapproximately 30% of the proteins.

[0141] A comparison of the galactosylation activity measured in a crudeextract of induced bacteria expressing the MGDG synthase A in thepresence and in the absence of DAG was carried out.

[0142] The galactosylation activity was measured by extraction of thelipids according to the method of Bligh and Dyer (see above) on 20 μl ofa bacterial culture induced at 28° C., in the absence of PG and possiblyplaced in the presence of 50 μg of DAG.

[0143] The results obtained appear in table I below: TABLE I Incubationof bacteria in 270 μM MGDG synthase activity (in nmole of of DAGgalactose incorporated per hour) yes 576 no 5

[0144] These results show that a considerable galactosylation activityis observed in this extract incubated with 270 μM of DAG, whereas thereis virtually no incorporation of galactose into the bacterial membranesincubated without the exogenous introduction of DAG.

[0145] Consequently, the bacterial membranes do not have a sufficientamount of endogenous DAG to carry out an isolated measurement of theactivity of the recombinant enzyme.

[0146] B—Treatment of the Bacterial Membranes with PLC Generates DAGAvailable for Measuring Galactosylation Activity

[0147] The galactosylation activity of the MGDG synthase was measuredafter synthesis of endogenous DAG by PLC or after addition of exogenousDAG. This activity was measured on 20 μl of the same bacterial cultureinduced at 28° C., in the presence or absence of 3 μl of PLC (2 000U/ml), of 50 μg of DAG (at 1 mg/ml and optionally in the presence of 1or 10 μl of CaCl₂.

[0148] The results are given in table II below: TABLE II DAG in μM PLCCaCl₂ in μM Galactose incorporated (nmol/hour) — — — 7 270 — — 30 — + —91 270 + — 99 270 + 33 118 270 + 330 109 — + 33 94 — + 330 97

[0149] These results show that treating the bacterial membranes with PLCmakes it possible to measure a galactosylation activity greater thanthat obtained after adding 270 μM of exogenous DAG.

[0150] The PLC therefore makes it possible to load the bacterialmembranes with DAG by hydrolysis of its phospholipids. The poorincorporation of galactose obtained when adding DAG is explained by thefact that a very small proportion of added DAG was able to penetrateinto the bacterial membranes in the absence of PLC, in order to act assubstrate for the MGDG synthase.

[0151] It is also important to note that the calcium, which is anactivator of PLCs, has no effect on the measured galactoseincorporation, suggesting that the concentration of Ca²⁺ of thebacterial suspension is sufficient to measure optimal activity of thePLC.

[0152] C—Incorporation of Radioactive Galactose into Membranes Enrichedin DAG

[0153] The incorporation of radioactive galactose into the lysedmembrane vesicles as described above in example 1, step B-3) wasmeasured and compared to the incorporation of radioactive galactose bybacteria induced to express MGDG synthase A, but not lysed. Thegalactosylation activity is measured on 150 μl of sample (as describedpreviously) in the presence or in the absence of 150 μg of PG (at 10mg/ml) and of 100 μg of exogenous DAG (at 1 mg/ml).

[0154] The results obtained are given in table III below: TABLE IIIGalactose incorporated (nmol/hour) Induced bacteria, Without exogenous+550 μM of exogenous treated with PLC introduction of DAG DAG and 667 μMof PG Not lysed 2 426 6 622 Lysed 3 172 8 663

[0155] These results show that the galactosylation activity measured onlysed bacteria without the addition of DAG or PG indicates that vesiclescontaining both the MGDG synthase and the DAG have been formed, and thatthe enzyme has conserved its catalytic capacity.

[0156] They also show that the activity of the lysed fractions isgreater than that of the non lysed fractions. This observation iscoherent with the MGDG synthase A being located on the inner face of thelipid bilayer of the bacterial membranes. Specifically, since the PLConly has access to the outer lipid bilayer, the fraction of MGDGsynthase located on the inner face can capture only a portion of theDAGs formed which have undergone transverse displacement from the outerleaflet to the inner leaflet of the membrane. The disruption of themembranes allows the formation of inverted and noninverted vesicles,but, in both cases, the MGDG synthase is on the face opposite most ofthe DAG. Fusion between inverted and noninverted vesicles may generate anew family of vesicles having, in the same membrane leaflet, the enzymeand the DAG. This type of vesicle may exhibit increased catalyticactivity (accessible substrate). If it is supposed that the generationof these three populations of vesicles, inverted, noninverted andhybrid, is equally probable, then the activity of the lysed samples isincreased by a factor of 1.33 compared to the non lysed samples, whichis observed in the present case.

[0157] D—Development of an Assay which can be Miniaturized

[0158] 1) Demonstration of the Role of PLC

[0159] A suspension of lysed bacteria containing membrane vesiclesenriched in MGDG synthase A and optionally in DAG by treatment with PLCis fractionated on a gradient of 35% Percoll (9 ml) as previouslydescribed. The fractions corresponding to the supernatant and to theinterface (S+SL: 6 ml) and also those corresponding to the pellet (P: 1ml) are collected. The galactosylation activity and the amounts ofproteins are measured on the various fractions sampled (S+SL=6 ml, P=1ml) and also of the fraction deposited (D of 6 ml).

[0160] The results obtained are given in table IV below: TABLE IV Totalactivity Specific (in nmol activity (nmol UDP-gal UDP-gal Proteins inincorporated/ incorporated/ Enrich- Fractions μg/ml hour) hour/mg) mentNo D 400 103  43 1 treatment S + SL 136 363 445 10.3 with PLC P 39  3 83 1.9 Treatment D 500 618 206 1 with PLC S + SL 880 11 462 2 169 10.5P 70  67 955 4.6

[0161] These results show that the membranes which have undergoneenrichment in DAG by treatment with PLC are capable of converting muchmore UDP-gal than the membranes not enriched in DAG, since they have notundergone any treatment with PLC.

[0162] Analysis of the fractions by acrylamide gel electrophoresis underdenaturing conditions shows enrichment for a polypeptide correspondingto MGDG synthase, from the supernatant to the pellet. The lowgalactosylation activity in the pellet (table IV) suggests an enrichmentof this fraction mainly in inclusion bodies (inactive enzyme). The S+SLfractions containing the MGDG synthase A associated with the membranesenriched in DAG were therefore selected in order to analyze the enzymeactivity of the MGDG synthase as a function of the criteria for validityof Michaelis-Menton enzymology.

[0163] 2) Analysis of the Enzyme Activity of the MGDG Synthase

[0164] The results are given in FIG. 1, which represents the initialrate (in nmol of galactose incorporated per hour) as a function of thenumber of μg of proteins/tube. The initial rates were determined bymeasuring the incorporation of galactose after 15 and 30 minutesincubation of various amounts of the membranes treated with PLC andpresent in the treated S+SL fractions (25, 50, 75 μg of proteins).

[0165] This FIG. 1 shows that the incorporation of galactose is a linearfunction of the amount of sample incubated in the presence of UDP-gal.The slope of the curve makes it possible to deduce an activity of 340nmol of galactose incorporated per hour and per mg of proteins.

[0166] The amount of DAG obtained after treatment with PLC isundetermined but the concentration of this substrate in the membranevesicles remains constant whatever the amount of proteins of the sample.In this respect, the surface concentration of DAG (in molar fraction orin mol of DAG per unit of membrane surface) and the molar concentrationof UDP-gal are constant. As a result, the initial reaction rate isdirectly proportional to the amount of proteins, showing the limitingnature of the amount of enzyme. The relationship of linearity is acondition of validation of conditions for Michaelis-Menton enzymologicalanalysis.

[0167] 3) Evaluation of the Amount of DAG Generated in the BacterialMembranes by Treatment with PLC

[0168] The activity (in number of nmol of galactoseincorporated/hour/mg) of 5 μg of membranes not treated with PLC andpurified on a Percoll gradient was measured for varying amounts of DAGintroduced (FIG. 2A). The initial rates were determined by measuring theincorporation of galactose after 15 and 30 minutes of incubation. Themeasurements were made for varying amounts of DAG introduced (12.5 μg;25 μg; 37.5 μg; per 300 μl of reaction medium).

[0169] The measurements make it possible to establish an invertedcoordinate plot of the Lineweaver and Burk type (FIG. 2B).

[0170] If it is considered that the measurements made on membranes nottreated with PLC constitute standard curves to estimate a DAG content, asample of 5 μg, the intrinsic activity of which is 340 nmol/h/mg, isequivalent to the same untreated sample to which 0.5 μg of DAG has beenadded.

[0171] It is therefore possible to deduce therefrom that the treatmentwith PLC makes it possible to obtain about 0.1 μg of DAG per μg ofproteins.

[0172] 4) Measurement of the Galactosylation Activity of MembranesEnriched in MGDG Synthase and in DAG Before and After Thawing.

[0173] The ability of membranes having undergone freezing for one day at−20° C. to incorporate galactose was measured and compared to that ofmembranes which had not undergone any freezing step.

[0174] No significant difference was observed between the two samples.

[0175] Consequently, this property makes it possible to prepare thesample of membranes and to manipulate it reproducibly in an automateddevice for screening molecules having an effect on MGDG synthaseactivity, which may comprise steps consisting of storing the sampleunder cold conditions, without loss of activity.

[0176] 5) Simplified Measurement of the Galactosylation ActivityContained in the Sample of Membranes Enriched in MGDG Synthase and inDAG

[0177] Membrane vesicles containing MGDG synthase enriched in DAG bytreatment with phospholipase C, and optionally purified, can thereforebe used to measure the production of MGDG in these same vesicles.

[0178] 50 μg of membrane vesicles (equivalent to 50 μg of MGDG synthaseand 50 μg of DAG) are suspended in an aqueous phase (250 mM KCl, 250 mMK₂HPO₄, 300 μl final volume, pH 7.8) contained in a tube (tube No. 1).

[0179] By way of comparison, a control tube (tube No. 2) was prepared:50 μg of MGDG synthase and 50 μg of DAG were suspended in the sameaqueous phase as that used in tube No. 1.

[0180] The reaction is initiated in each tube by adding 10 μl of UDP-galradiolabeled with ¹⁴C on the galactose, and then stopped after 30minutes by cooling to 4° C.

[0181] The reaction medium of tube No. 1 is then centrifuged at 13 000rpm for 10 minutes, rinsed several times with 500 pi of water, thencentrifuged again at 13 000 rpm for 10 minutes.

[0182] Tube No. 2 was treated using the conventional method of lipidextraction with organic solvents, as described previously.

[0183] The radioactivity recovered in the centrifugation pellet (tubeNo. 1) was measured using a radioactive counter and was 4 026 dpm.

[0184] The radioactivity measured after extraction of the lipids in tubeNo. 2 was measured in the same way and was 4 136 dpm.

[0185] Consequently, the radioactivity measured for each of the tubes isnot significantly different and makes it possible to validate the methodof measuring the MGDG synthase activity in accordance with theinvention.

[0186] The sample of membrane vesicles prepared in accordance with theinvention contains the enzyme and its hydrophobic cosubstrate underconditions which correspond to measurement of the enzymological activityby the Michaelis method. In addition, this sample allows simple andminiaturizable measurement of the galactosylation activity bycentrifugation and, by extension, by filtration.

[0187] VI—Conclusion

[0188] This example demonstrates that it is possible to use membranefractions derived from a culture of cells expressing a recombinant MGDGsynthase to measure a galactosylation activity respectingMichaelis-Menton laws, and the procedure for the measurement of whichcan be miniaturized on microplates.

[0189] It has also been demonstrated that the incorporated radioactivitycan be simply recovered in the aggregates of the reaction medium,collected by centrifugation. Thus, the MGDG generated by this systemaccumulates in the membranes that a system of measurement bymicrofiltration is sufficient to measure, making it possible to use thisenzyme assay for high throughput screenings (HTS) of active molecules.In particular, it is now possible to screen a “chimiotheque” [chemicallibrary] by this method in order to select novel molecules withherbicidal potential. The inhibitors specific for MGDG synthase A thusproduced will, moreover, be powerful pharmacological tools for allowingphysiological studies of galactolipid synthesis.

EXAMPLE 3 Demonstration of MGDG Synthesis in an Apicomplexan Parasite

[0190] The aim of this example is to demonstrate the presence of MGDG inan apicomplexan parasite, Toxoplasma gondii and that, consequently, theMGDG synthase which serves as a target to search for a molecule withantiparasitic properties clearly exists in apicomplexans.

[0191] 2×10⁸ cells of Toxoplasma gondii, in the form of tachyzoites, aresuspended in 0.1 ml of a 1:10 mixture of 10 mM3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.8 and 1 mMdithiothreitol (DTT), containing 2% (w/v) glycerol and 50 mM of KCl, andthen incubated for 30 min in the presence of 4 μCi of UDP-[³H]-galactose(7.5 nmol).

[0192] The glycolipids are extracted according to the method of Blighand Dyer, 1959 (mentioned above), then analyzed by thin layerchromatography (TLC) on 60μ silica gel plates resolved with a 65/25/4(v/v) chloroform/methanol/water mixture, in the presence of controllipids (MGDG; bovine brain monogalactosyl cerebroside (MGCB);digalactosyldiacylglycerol (DGDG); trigalactosyldiacylglycerol (triGDG)and tetragalactosyldiacylglycerol (tetraGDG)).

[0193] The radioactivity of the labeled lipid is then detected using aTLC-analyzer device (LB2842 automatic TLC scanner).

[0194] The results obtained are given in FIG. 3, which illustrates theamount of labeled galactose (cpm) incorporated by the Toxoplasma gondiicells as a function of migration in centimeters.

[0195] On this figure, it is possible to see 3 first peaks which migrateto the same degree as the MGCB, whereas the last peak migrates to thesame degree as the MGDG.

[0196] After migration, the lipids are visualized by spraying, onto thesilica gel plates, a solution comprising 0.2% of orcinol and 75% ofsulfuric acid, and then heating at a temperature of 100° C. for 15minutes (results not given).

[0197] The peak corresponding to the MGDG disappears after alkalihydrolysis for 3 hours with 0.1 N potassium hydroxide in awater/methanol mixture.

[0198] Complete identification of the lipids is carried out afterhydrolysis of the polar head with α-galactosidase from green coffeebeans and β-galactosidase from bovine testes, and deacylation by alkalihydrolysis under gentle conditions (0.1 N KOH in a water/ethanol mixturefor 3 hours).

[0199] Peak 4 is sensitive to hydrolysis with β-galactosidase, whichshows that the galactose is clearly linked in the beta position, as forMGDG.

[0200] Moreover, after alkali hydrolysis, peak 4 disappears, whichdemonstrates that the lipid present in the Toxoplasma gondii membraneclearly contains half diacylglycerol.

[0201] This experiment demonstrates the existence of MGDG in themembrane of Toxoplasma gondii tachyzoites and confirms that anapplication of the search for inhibitors of plant MGDG synthase is theidentification of anti-apicomplexan parasite agents.

1. Plasma membrane fractions from prokaryotic cells or eukaryoticanimals, consisting of a lipid leaflet containing at least onerecombinant MGDG synthase, characterized in that said fractions containat least 1% by weight of DAG relative to the total weight of protein,and in that they are in the form of spherical vesicles.
 2. The fractionsas claimed in claim 1, characterized in that the MGDG synthase/DAG molarratio is less than
 10. 3. The fractions as claimed in claim 2,characterized in that the MGDG synthase/DAG molar ratio is less than0.12.
 4. The fractions as claimed in any one of the preceding claims,characterized in that they are in the form of spherical membranevesicles made up of a lipid bilayer.
 5. The fractions as claimed in anyone of the preceding claims, characterized in that the membrane vesiclesare between 0.1 μm and 10 μm in size.
 6. The fractions as claimed inclaim 4 or 5, characterized in that the MGDG synthase is located on theinner face of the lipid bilayer.
 7. The fractions as claimed in any oneof claims 4 to 6, characterized in that the vesicles are in the form ofnoninverted, inverted or hybrid vesicles.
 8. The fractions as claimed inclaim 7, characterized in that they are in the form of hybrid vesiclescomprising the MGDG synthase and the DAG in the same lipid leaflet.
 9. Amethod of preparing membrane fractions as defined in any one of thepreceding claims, characterized in that it consists: in a first step, intransforming prokaryotic cells or eukaryotic animals with a constructcontaining the gene encoding a plant MGDG synthase, in a second step, inculturing said cells in a culture medium which promotes proteinsynthesis, so as to induce the synthesis of MGDG synthase by said cells,in a third step, in incubating the cells cultured in the preceding stepin a reaction medium containing at least one phospholipase C, and then,in a fourth step, in fractionating the cells thus enriched in DAG so asto obtain membrane fractions in the form of spherical vesiclescontaining at least one recombinant MGDG synthase and at least 1% byweight of DAG relative to the total weight of proteins of said membranefractions.
 10. The method as claimed in claim 9, characterized in thatthe prokaryotic cells are bacteria.
 11. The method as claimed in claim 9or 10, characterized in that a Bacillus cereus phospholipase C is used.12. The method as claimed in any one of claims 9 to 11, characterized inthat the phospholipase C is used at a concentration between 1 U and 20 Uper ml of reaction medium.
 13. The method as claimed in any one ofclaims 9 to 12, characterized in that the fractionation of the membranesis carried out by mechanical shock, thermal shock, osmotic shock,electric shock or by physical shock.
 14. The use of the membranefractions as defined in any one of claims 1 to 8, for selecting and/orscreening molecules having an effect on MGDG synthase activity.
 15. Theuse as claimed in claim 14, for selecting and/or screening moleculesinhibiting MGDG synthase activity, able to be used as active principlesagainst parasites or as herbicides.
 16. A method of selecting and/orscreening molecules having an effect on MGDG synthase activity,characterized in that it comprises: a step comprising incubation of thetest substance(s) with a sufficient amount of membrane fractions asdefined in any one of claims 1 to 8 and of radiolabeled UDP-galactose inan aqueous phase having a pH of between 4 and 11, a step comprisingwashing of the membrane fractions, a step comprising separation of themembrane fractions, then a step comprising determination of the MGDGsynthase activity.
 17. The method as claimed in claim 16, characterizedin that the aqueous incubation phase contains a buffer and has a pH ofbetween 6 and
 8. 18. The method as claimed in claim 16 or 17,characterized in that the amount of UDP-galactose is between 0.1 and 10nmol per μl of reaction medium.
 19. The method as claimed in any one ofclaims 16 to 18, characterized in that the step comprising separation ofthe membrane fractions is carried out by centrifugation or byfiltration.
 20. The method as claimed in any one of claims 16 to 19,characterized in that the determination of the MGDG synthase activity iscarried out by measuring the amount of radiolabeled galactoseincorporated into the membrane fractions.
 21. Microtitration platescomprising a multitude of wells, characterized in that the bottom of thewells consists of a filter and in that said wells contain membranefractions as defined in any one of claims 1 to 8.